Human cytomegalovirus (HCMV) like other members of the herpes virus group encodes a cysteine protease (deubiquitylase, DUB) with specificity to cleave the isopeptide bond between ubiquitin and the protein it is attached to. The catalytic domain is at the amino end of a large structural protein of the virion tegument (HCMV pUL48), is about 400 amino acids long, and is conserved among herpes viruses. Inactivating the DUB results in lower virus yields in cell culture and reduced pathology in animal models. The function of this enzyme remains elusive and a molecular target substrate common to all herpes viruses has yet to be identified. We have detected ubiquitylated proteins in virions of HCMV and propose here to recover and identify them, with the longer-range aim of understanding their purpose in the virion, whether they are substrates of the viral DUB that is incorporated into the virion with them, and what insight they may provide into mechanisms of herpes virus tegumentation or envelopment or new antiviral strategies. Our approach takes advantage of viruses designed to express a 6His-tagged form of ubiquitin that will be conjugated to target proteins and enhance their detection, recovery, and analysis. A mutant virus additionally expressing an inactive DUB has also been constructed and increases the extent to which the virion proteins of interest are ubiquitylated. These 6His-tagged virion proteins will be recovered from purified extracellular particles by immobilized metal affinity chromatography, and analyzed by mass spectrometry to identify the presence of ubiquitin and its site of attachment. Cytomegalovirus can be debilitating if not life threatening in immunocompromised patients, particularly those having undergone organ transplantation or those with AIDS or cancer. Congenital cytomegalovirus infections can be passed to a fetus or newborn from the mother and can be sexually transmitted between adults. There is an increasing need for effective drugs to treat these infections, and new antiviral targets such as the herpes virus DUB are of interest in that regard.